Characterization of the IncA/C plasmid pCC416 encoding VIM-4 and CMY-4 beta-lactamases

Objectives: To characterize the antibiotic resistance regions of pCC416, a VIM-4- and CMY-4-encoding plasmid from clinical enterobacteria, and to elucidate its relation with the CMY-encoding plasmids widely diffused in Salmonella. Methods: The enterobacterial multiresistant plasmid pCC416 was derived from an Escherichia coli transconjugant and characterized. Conventional and long-range PCR assays were performed using primers specific for VIM-4- and CMY-4-encoding segments of pCC416. Amplicons were characterized by sequencing. bla(VIM-4), bla(CMY-4) and Intl1-specific probes were prepared from PCR products and used for the identification of various pCC416 clones. VIM- and CMY-positive BamHI and Sau3AI fragments of pCC416 were cloned into pACYC184 and their sequences were determined by gene walking. Results: The pCC416 plasmid contained two distinct resistant loci carrying beta-lactamase genes. The bla(VIM-4) gene was part of an integron located in a complex, multidrug-resistant region of novel structure, interspersed with mobile elements or remnants thereof and being similar to various regions of other resistance plasmids. Nevertheless, a region in the 3' end of this structure resembled the respective region found in a CMY-2-encoding plasmid from Salmonella. The bla(CMY-4) gene was identified within an 11.3 kb region also related to the CMY-2-encoding plasmids. Conclusions: pCC416 probably evolved from an IncA/C-2, CMY-encoding plasmid through acquisition of a VIM-encoding In4-type integron providing an example of accretion of resistance determinants in a single replicon.